Background: β-lactoglobulin hydrolysates (BLGH) have shown antioxidant, antihypertensive, antimicrobial, and opioid activity. In the current study, an innovative combination of high hydrostatic pressure and enzymatic hydrolysis (HHP–EH) was used to increase the yield of short bioactive peptides, and evaluate the anti-inflammatory and antioxidant properties of the BLGH produced by the HHP–EH process. Method: BLG was enzymatically hydrolyzed by different proteases at an enzyme-to-substrate ratio of 1:100 under HHP (100 MPa) and compared with hydrolysates obtained under atmospheric pressure (AP-EH at 0.1 MPa). The degree of hydrolysis (DH), molecular weight distribution, and the antioxidant and anti-inflammatory properties of hydrolysates in chemical and cellular models were evaluated. Results: BLGH obtained under HHP–EH showed higher DH than the hydrolysates obtained under AP-EH. Free radical scavenging and the reducing capacity were also significantly stronger in HHP-BLGH compared to AP-BLGH. The BLGH produced by alcalase (Alc) (BLG-Alc) showed significantly higher antioxidant properties among the six enzymes examined in this study. The anti-inflammatory properties of BLG-HHP-Alc were observed in lipopolysaccharide-stimulated macrophage cells by a lower level of nitric oxide production and the suppression of the synthesis of pro-inflammatory cytokines. Peptide sequencing revealed that 38% of the amino acids in BLG-HHP-Alc are hydrophobic and aromatic residues, which contribute to its anti-inflammatory properties. Conclusions: Enzymatic hydrolysis of BLG under HHP produces a higher yield of short bioactive peptides with potential antioxidant and anti-inflammatory effects. Whey is a byproduct of cheese manufacturing, with a broad range of applications in the food and nutraceutical industries . The maiAlthough whey proteins have nutritional and functional benefits, their utilization is restricted mainly due to the Cow’s Milk Protein Allergy (CMPA), which is the most prevalent food allergy, particularly in infants . BLG is A major challenge in protein hydrolysis is the cost of the enzyme used. The simultaneous use of high hydrostatic pressure (HHP) and enzymatic hydrolysis  improves the activity of proteolytic enzymes and eliminates the need for pre-treatments, resulting in a lower processing cost . FurtherThe main objective of this research was to optimize the HHP–EH process to produce BLG hydrolysates with antioxidant and anti-inflammatory properties. Various enzymes, and pressures were used to determine the optimal conditions for BLG hydrolysis with the highest degree of hydrolysis. Enzymatic hydrolysis was also performed at atmospheric pressure (AP) for comparison. The BLG hydrolysates were then tested for antioxidant and anti-inflammatory properties, as preliminary tests for potential bioactivity. The amino acid sequences of the peptides in effective BLG hydrolysates were also identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), to determine the effective amino acids contributing to the antioxidant and anti-inflammatory properties.The BLG was hydrolysed by neutrase (Neu), alcalase (Alc), savinase (Sav), elastase (Ela), thermolysin (Ther), and trypsin (Try), at an enzyme-to-substrate (E:S) ratio of 1:100, and under the optimal conditions for each enzyme . The degThe MALDI-TOF spectra of BLG hydrolysates displayed a wide molecular weight range from <500 to 3000 Da. Regardless of the pressure treatment, the BLG hydrolysates showed an iron chelating capacity (42–58% at 500 μg/mL) comparable to EDTA (58% at 10 μg/mL) B in a doThe reducing capacity of BLG hydrolysates was determined by FRAP assay, and expressed as a μM Trolox equivalent C. All thThe BLG hydrolysates produced by Alc resulted in the highest degree of hydrolysis, and showed the best antioxidant properties among the enzymes examined in this study. Therefore, Alc was used to determine the effect of HHP and AP on the anti-inflammatory properties of BLG.The results obtained from 3--2,5-diphenyltetrazolium bromide (MTT) assay, as presented in The gene expression of pro-inflammatory cytokines (tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β)) in LPS-stimulated RAW264.7 macrophage cells was studied using BLG-Alc hydrolysates produced under HHP and AP conditions. The BLG-Alc hydrolysate significantly suppressed the gene expression of pro-inflammatory cytokines (TNF-α and IL-1β) in LPS-stimulated RAW264.7 macrophage cells C, and shThe BLG hydrolysate produced by Alc was subjected to peptide sequencing using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and the results are shown in In the last decades, whey proteins have been used to produce high quality protein hydrolysates with improved bioavailability, bioactive functionality, and lower allergenicity. Previous studies have reported the effects of different treatments on whey protein hydrolysis ,19,27. IHigher pressure levels—up to 100 MPa—alter the protein volume (compressibility) and hydration shell surrounding the protein molecule by changing the exposure of functional groups, leading to the higher susceptibility of protein to enzymatic catalysis ,22,31,32The SDS-PAGE pattern  of hydroCompared to AP-hydrolysates, the free radical scavenging and reducing capacities of HHP-BLG hydrolysates were significantly stronger . HoweverThe MTT assay was used to demonstrate the effect of BLG hydrolysates on RAW264.7 macrophage cell viability. After a 24 h incubation, both BLG-HHP-Alc and BLG-AP-Alc showed no significant variation in cell viability A. The deNitric oxide (NO) production is an inflammatory response to oxidative stress in cells. The overproduction of NO has been found to play a role in certain diseases, such as diabetes mellitus, neurodegenerative disorders, and other inflammatory conditions. Antioxidant peptides can suppress oxidative signaling, and therefore alleviate inflammatory responses. The LPS-stimulated macrophage cells produced NO to a degree several times higher than that of untreated cells. We evaluated the NO production of the LPS-stimulated macrophage cells in the presence of HHP- and AP-BLG hydrolysates. Cells incubated with BLG-HHP-Alc produced significantly lower NO compared to cells incubated with BLG-AP-Alc B. This rDifferent mechanisms have been proposed for the inhibition of LPS-induced effects on macrophage cell lines by proteins and peptides . These mIn addition to NO, LPS-stimulated RAW264.7 macrophage cells can release large amounts of pro-inflammatory cytokines, such as TNF-α and IL-1β. These cytokines can further activate macrophage cells, and stimulate the production of other inflammatory cytokines, contributing to various pathophysiological conditions . DownregThe antioxidant and anti-inflammatory peptides from the BLG-Alc hydrolysate were subjected to LC-MS/MS analysis. Eight peptides derived from BLG were characterized. The bioactivity of peptides can be influenced by various factors, including peptide size, composition, and the positioning of the amino acids in the sequence . The occTherefore, our findings showed that HHP–EH was able to produce short peptides from BLG protein with a high antioxidant and anti-inflammatory activity. While extreme physical and chemical conditions increase the DH, these harsh conditions reduce the biological activity of the peptides. Thus, HHP–EH improves hydrolysis efficiency without compromising the peptides’ bioactivity. Furthermore, HHP–EH is a very simple and economic alternative to the conventional methods of protein hydrolysis, with a lower risk of contamination and a higher nutritional value of the product.2. All the chemicals and reagents were of analytical grade, and obtained from commercial sources.The β-lactoglobulin (85%) was purchased from Sigma-Aldrich Inc . The enzymes (Neutrase (Neu), Alcalase (Alc), Savinase (Sav), Elastase (Ela), Thermolysin (Ther), and Trypsin (Try)) were purchased from Sigma-Aldrich, MP Biomedicals , Novozymes , and Fisher Scientific . The mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection , and cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS), penicillin/streptomycin/fungizone, and other required ingredients. The cell cultures were maintained at 37 °C in a humidified atmosphere of 5% COA potable high-hydrostatic pressure machine  was used for the HHP process, under 100 ± 2 MPa pressures at 37 °C. The vessel was filled with ultrapure water as a fluid of low compressibility. The protein–enzyme mixture, placed in a sealed pouch, was kept in a water bath to maintain a constant vessel temperature during the high hydrostatic pressure treatment.g, 4 °C, 30 min) to remove the insoluble parts. The supernatants and pellets were separated and stored at −20 °C prior to analysis. The protein content of the BLG hydrolysates was determined using the Bio Rad Proteins Assay . The results were expressed as mg protein/g dry powder.The β-lactoglobulin was dissolved in an appropriate buffer, at the protein concentration of 10 mg/mL. The BLG solution was hydrolyzed using six different enzymes at an enzyme-to-substrate (E:S) ratio of 1:100, under HHP at 100 MPa for 2 h at 37 °C. For comparison, hydrolysis was also performed under atmospheric pressure  for 2 h at 37 °C. The above procedures were repeated in the absence of enzymes, to act as experimental controls. After the digestion, the enzymes were inactivated by heat treatment , and centrifuged at  fluorometric assay [tot is the total amount of amino groups present in totally hydrolyzed BLG with 6 N HCl at 110 °C for 24 h.The degree of hydrolysis was determined by the quantification of the free amino groups released during the hydrolysis reaction by the ic assay . BrieflyThe gel electrophoresis was performed according to the method of Schaegger, in Mini-PROTEAN Tris-Trycine Precast Gels in Tris-Trycine-SDS buffer  with 12% polyacrylamide gels . The PreMALDI-TOF-MS analysis was performed at Alberta Proteomics and Mass Spectrometry . One µL of each sample was mixed with 1 µL of sinapic acid (10 mg/mL in 50% acetonitrile/water + 0.1% trifluoroacetic acid), and then spotted onto a stainless steel target plate and allowed to air dry. All mass spectra were obtained using a Bruker Ultraflex MALDI-ToF/ToF . The ions were analyzed in the positive mode, and an external calibration was performed by use of a standard protein mixture.s and Ac represent the absorbances of the sample and the control (everything except the protein hydrolysate), respectively.A DPPH free radical was used to determine the free radical scavenging activity of BLG hydrolysates at different concentrations  . Glutath2 was added to 0.5 mL of BLG hydrolysates , and the reaction was then initiated by the addition of 1 mL ferrozine (0.5 mM). The mixture was vortexed, and left standing at 23 °C for 10 min. Ferrozine- Fe2Cl is a pink chromophore that absorbs strongly at 562 nm [s and Bc represent the absorbance of the sample and the control (deionised water instead of hydrolysates), respectively.One mL of 20 mM FeClt 562 nm . EDTA, aThe antioxidant capacity of the samples  was estimated according to the procedure described by Fogarasi et al. . Trolox The Raw264.7 cell proliferation was evaluated using the MTT assay [2−) in the culture media [v/v) with Griess Reagent in 96-well plates, followed by 15 min of incubation at an ambient temperature. The absorbance was recorded at 540 nm using a UV-visible spectrophotometer . Cells grown in media, without any treatment, were taken as a negative control. The positive control cells were only stimulated with LPS (without BLG hydrolysate treatment). Sodium nitrite at different concentrations (0.1–100 μM) was used to make a standard curve (R2 = 0.9998).The effect of the BLG hydrolysates on NO production by stimulated macrophages was evaluated by a determination of the accumulated nitrite  in the presence or absence of Alc-treated samples (10 µg/mL). Cells incubated in media served as the negative control. Cells treated only with LPS (1 µg/mL) were taken as the positive control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. After 24 h, the RNA was extracted from the cells using the TRIzol-chloroform method. The RNA’s purity was measured by a spectrophotometric method. The total RNA was reverse-transcripted into cDNA (in 20 µL of RT-reaction mixture) using the 5X All-In-One RT MasterMix for RT-PCR. The quantitative real-time-PCR was performed in a Quantstudio III . The 10 µL reaction mixture consisted of 2.5 ng cDNA, 300 nM of each primer , and 5 µL of EvaGreen 2X qPCR MasterMix-Low Rox. Water was used as a control. The reaction conditions were initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. Each mRNA expression was normalized against GAPDH mRNA (ΔΔCt-method), and all data are presented as a fold change against the unstimulated control [RAW264.7 cells were seeded in a six-well plate at 5 × 10 control .18 PepMap 100 Nano-Precolumn™ , and a nano analytical column . Desalting on the peptide trap was achieved by flushing the trap with 1% acetonitrile, and 0.1% formic acid in water at a flow rate of 10 µL/min for 1 to 3 min. The peptides were separated with a gradient of 1–60% solvent B  over 55 min at a flow rate of 350 nL/min. The column was connected to a q-Tof premier  for ESI-MS/MS analysis. The obtained MS/MS data were analyzed through proteomic software called Mascot . The settings for the UniProt database search were as follows: parent ion and MS/MS tolerance were set to 0.1 Da and 0.2 Da, respectively, and up to two missed cleavages were selected; oxidation on methionine was selected as a variable modification. The confidence of positive protein identification was judged by high protein and peptide scores in the search results. A manual inspection of the original MS/MS spectra was often performed to make sure major peaks in the MS/MS spectra were matched and explained.The hydrolysate was subjected to LC-MS/MS analysis on a nano UPLC , coupled with a q-Tof premier mass spectrometer . Five µL of the peptide mixture was loaded onto a nanoAcquity UPLC system, with a Cp < 0.05) was evaluated by the least significant difference test.All experiments were performed at least in three independent trials, and the results were reported as mean ± SD. The results were subjected to the analysis of variance using the ANOVA procedure of SAS , and the statistical significance of differences  were obtained when the BLG was hydrolysed using the HHP–EH method. These small peptides showed an excellent free radical scavenging capacity, and iron chelation and reducing power. The HHP-BLG hydrolysates were cyto-compatible, and did not adversely influence macrophage cell growth. A significant reduction in NO level after pre-incubation with BLG hydrolysates indicates the anti-inflammatory properties of hydrolysates. This was also evident by the significant decrease in the expression of pro-inflammatory cytokines after the cells were treated with HHP-BLG-Alc hydrolysate. Accordingly, HHP–EH is a promising technology to produce shorter peptides from various protein sources under a mild hydrolysis condition. The structural and functional properties of hydrolysates obtained from the HHP–EH digestion of whey proteins are currently being studied in our lab.


The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules.Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC Hapten B had been synthesized in our previous work . The synFigure 12O. The resultant solution was cooled in an ice bath and the pH was adjusted to 4.5 with HCl (1M). Then, 320 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added (with stirring) to the above solution in small portions. The pH of this reaction mixture was kept at 4.5~5.0 (with HCl) throughout the addition of EDC and the mixture was then stirred at 4 °C over night. The conjugation mixture was dialyzed against 10 mM PBS (4 × 5 L) at 4 °C for 72 h, and diluted to 1.0 mg/mL with PBS and stored at –20 °C until used. As with Hapten B, HA-245 also has a secondary amine and a carboxyl group, thus HA-245 was conjugated to carriers (BSA or OVA) using the same one-step EDC method as that for preparation of Hapten B-protein conjugates instead of the two-step activated ester method to avoid lactamization. Briefly, 0.12 mmol of hapten and 67 mg of BSA (or 45 mg of OVA) were added in 10 mL of HAnimal manipulations were performed in compliance with the Regulation Guideline for Experimental Animals issued by the Ministry of Science and Technology of China  2014-0136; Approval date: 16 May 2017; Ethics committee: Guangdong Provincial Department of Science and Technology). g, 30 min) by centrifugation. The obtained antisera were tested for their anti-hapten antibody titer and binding properties by non-competitive indirect ELISA and ic-ELISA  in 0.2 mL of emulsion (a 1:1 mixture of immunogen (1 mg/mL) and complete Freund’s adjuvant). The booster immunizations were given 3 times using the same dose of immunogen emulsified in the incomplete Freund’s adjuvant were given at two-week intervals. One week after the last immunization, the mice were tail-bled and the blood samples could coagulate at 37 ℃ for three hours. Then, the antisera  was separated  . For furThe specificity of the antibody was assessed by testing cross-reactivities (CRs) with some compounds (cross-reactants) which are structurally or functionally related with target molecule using ic-ELISA. The CR was calculated using the following equation:4  to secondary amine by NaBH4 . Hapten 4 . It was 50 of 0.55 mg/L which was improved ~100-fold compared to that of the anti-HA-245#2@HA-245-OVA homologous combination. Therefore, the hapten design strategy proposed above proves to be effective for HA. Furthermore, the NO.2 mice immunized by immunogen HA-245-BSA was chosen as the donor of the spleen cells for cell fusion. It was found that five wells among 480 wells were determined to have positive hybridoma cells which were subcloned five times using the limiting dilution method. One hybridoma (3E9) secreting Mab showing affinity to the HA was obtained. The efficacy of two immunogens  were assessed by immunization of groups of n = 3 Balb/c mice. Each immunogen was administered on weeks 0, 2, 4 and 6 with bleeds taken one week following the last immunization. The anti-HA antiserum titers and binding properties were determined by ELISA using Hapten B-OVA and HA-245-OVA as coating antigen, respectively. As shown in 50 (the HA dose that produce 50% decrease in Amax) and ratio of Amax to IC50 (Amax/IC50) were compared. The highest ratio of Amax to IC50 indicated the optimal condition. As shown in 43– strength (5–40 mmol/L), and pH values (5.6–8.5) were used as a working buffer. As a result, PBS with a PO43– strength of 20 mmol/L and a pH of 6.8 were confirmed to be the optimal working buffer. Under the optimal condition, the standard ic-ELISA inhibition curve for HA was constructed  were 0.21 mg/L and 0.06 mg/L, respectively. The obtained anti-HA Mab was further used to develop an ic-ELISA for HA. To achieve optimal assay performance, some key parameters including the amount of antibody and coating antigen, pH and ionic strength of the assay buffer were optimized. Under each condition, ic-ELISA inhibition curves for HA were developed, and then several index including Amax , ICstructed . The IC5The cross-reactivity (CR) test was carried out by measuring CRs between anti-HA Mab and several HA or hapten HA-245 related compounds through ic-ELISA. It was shown in g for 10 min, after removing the fat layer, the supernatant was ready for ic-ELISA analysis. Then, 1 mL of red wine or soy sauce was diluted 10-fold in PBS  and subjected to the ic-ELISA analysis. Further, 1 g of yoghourt and 9 mL of PBS  were mixed and vibrated for 1 min. The mixture was centrifuged at 3000× g for 10 min, the supernatant was ready for ic-ELISA analysis.The high levels of HA usually contaminate seafood, especially, Scombroid fish such as tuna, mackerel, bonito, and saury that are rich in free histidine in their muscle ,15. MeanThe recovery tests for the intra-assay and inter-assay were performed by analyzing saury, red wine, soy sauce and yoghurt samples spiked with four levels of HA . As shown in Sixteen blind samples were purchased from local supermarkets, then their HA contents were analyzed by both this ic-ELISA and LC-MS/MS which was established in our previous work , respectThe incorporation of a linker ending with a reactive group when designing hapten was generally preferred. According to conventional hapten design criteria, a linear aliphatic linker with a length of 2 to 6 carbon atoms was preferable for an optimal hapten, since it caused minimum physico-chemical and antigenic interference over the hapten. To produce the desired antibodies, much more attention has been invested on the effect of the hapten linker site on the binding properties (affinity and specificity) of resultant antibodies, since the linker site would mold the final conformation of the conjugate and thus settle the specific moieties that could be accessible for binding. For example, Mercader et al. produced a high-affinity anti-pyraclostrobin antibody by synthesizing a series of anti-pyraclostrobin haptens with the same aliphatic linkers located at different sites, and found that the lower titers and affinities of one set of antibodies were most likely due to the conformational effects of the linker on the immunizing bioconjugate . Wang etNonetheless, it should be noted that the incorporation of phenyl-contained linker should induce a minimum possible alteration in the electronic and conformational properties of the target molecules. As seen with the anti-NPHA hapten  designed to have an aliphatic linker in the same position as that in immunizing hapten (HA-245) was used as linker-heterologous coating hapten, and significantly improved the binding abilities with HA were observed for these antibodies.In summary, this investigation demonstrated the effectiveness of modulating the linker composition of immunizing haptens from conventionally preferred linear aliphatic linker to a phenyl-contained linker in improving the quality of antibodies against HA. However, for this hapten design strategy, it should be noted: (i) The incorporation of phenyl-contained linker should not induce physico-chemical and antigenic interference over the hapten as much as possible, which could be assessed by the computer-assisted molecular modeling; (ii) when developing the immunoassay, it is necessary to eliminate the linker recognition by using linker-heterologous haptens which have a phenyl-free linker as competitive haptens. This work realized derivatization-free immunoassay for HA and provided a valuable guidance for hapten design and development of immunoassays for other small molecules.


To improve the mechanical strength and antipollution properties of membranes, this research presents a facile method to prepare crosslinked polyacrylonitrile (PAN) membranes. This was achieved firstly by radical copolymerization with acrylonitrile, allyl methacrylate and sulfobetaine methacrylamide. Then, the copolymer was crosslinked by a thiol-ene click reaction under UV irradiation. Finally, the crosslinked membranes were prepared by traditional immersion precipitation phase inversion. These prepared membranes showed excellent water-pressure resistance and solvent swelling, owing to their crosslinked structure. This research will help in preparing crosslinked membranes through facile crosslinking under mild reaction conditions. The betaine structure also considerably improved the antifouling properties of the membranes. Membrane technology develops rapidly, and has been widely applied in many fields such as water treatment, chemical reagent distillation, gas separation and protein purification ,2,3. ForMembrane processes such as ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) are the most effective strategies for freshwater augmentation. However, traditional membrane processes operate under high pressure, and therefore, the membranes used should have good mechanical strength. At the same time, the thickness of membranes can be decreased for better water flux if the membranes’ mechanical strength is high enough. On the other hand, for stable structures, low swelling is very important to treat wastewater with high concentrations of organic compounds. Two measures have been taken to enhance the mechanical strength of membranes: using high strength materials such as polysulfides, aromatic polymers, or polyamides, and preparing composite membranes. However, both methods increase the water-treatment cost ,11.Here, we present a facile crosslinking technique under UV irradiation by using the thiol-ene click reaction to prepare a crosslinked PAN membrane. This crosslinking process can be completed within 1 min under UV irradiation ,13,14. TMη = 8.0 × 104 g/mol) dimethyl-(3-sulfopropyl) ammonium hydroxide inner salt  was synthesized according to a published procedure . The PAN4 g/mol) .To prepare crosslinked membranes, the P(AN-co-SBMAA-co-AMA) with pendent unsaturated allyl groups was synthesized firstly via the conventional free-radical copolymerization of the AN, AMA and SBMAA monomers, using AIBN as the initiator, and CYS as the chain transfer agent to avoid gel formation . AN (21.PAN-based crosslinked membranes were prepared by the immersion precipitation phase inversion (IPPI) technique. Firstly, P(AN-co-SBMAA-co-AMA) copolymers with pendent allyl groups were dissolved in DMSO at 60 °C to make an 8 wt % homogeneous casting solution. Then, benzoin methyl ether (DMPA) as a photo initiator (2.5 wt %) and DMDO as a crosslinker were added, and the solution was stirred for 15 min at room temperature, then left for 1 h to allow the release of bubbles. The casting solution was spread onto a clean glass plate using a casting knife with a 200 μm gate opening. Next, the nascent membrane was immediately cured with a 300 W high-pressure mercury lamp at a distance of 50 cm for 60 s. Finally, the glass plate was immersed into a coagulation bath of deionized water for 30 min. After peeling off from the glass plate, the membranes were rinsed with ethanol to remove the residual crosslinker, and kept in water until use. To evaluate the impact of crosslinking density on the membranes’ structure and properties, different concentrations of the DMDO  were applied onto the copolymers. The resulting crosslinked membranes were designated as CM-0, CM-0.4, CM-0.8 and CM-1.2, respectively.1H-NMR spectrometer using DMSO-d6 as the solvent.The P(AN-co-SBMAA-co-AMA) copolymer composition was checked, by a Nicolet 6700 TA  Fourier-transform infrared spectroscopy (FT-IR) spectrometer, with a polymer sample mixed with potassium bromide powder and pressed into a tablet. The copolymer’s chemical structure was further analyzed by an Avance III 500 MHz Bruker  0), and dried in a vacuum oven until the weights stabilized. Then, the membranes were weighed, and the weights were regarded as their initial dry matter (m0). Subsequently, the membranes were directly immersed into 30 mL of DMSO at 60 °C for 24 h. Next, the membranes were blotted by a filter paper and their dimensions were measured (Sf). Finally, the membranes were gently rinsed with ethanol for three days, and dried in a vacuum oven until the constant weight (fm) was reached. The gel fraction (GF) and dimensional change (ΔS) were calculated by the following equations:The crosslinking density of the membranes was evaluated by testing their swelling, water pressure resistance and mechanical properties. For the swelling test, each PAN-based crosslinked membrane was cut into five samples of dimension 2 cm × 2 cm . Each RP was obtained by performing three repeated experiments.The mechanical properties of the crosslinked membranes were measured by a universal testing machine to determine the tensile strength and Young’s modulus in the swollen state at room temperature. Dumbbell-shaped strips (6 mm × 117 mm) were prepared from each sample and soaked in deionized water before testing. Each test was carried out six times. Only the mechanical properties of the swelling membranes were assessed, because the dried membranes were fragile and folded.The membrane morphologies were observed by a Carl Zeiss AG supra 55  field-emission scanning electron microscope (SEM) operating at an accelerating voltage of 5 keV. Prior to SEM analysis, the membranes were affixed to a standard sample stub by double-sided carbon conductive tape, and a thin layer of gold was sputtered onto the sample surface.Jw is the water flux (L/m2h), V is the volume of the permeated pure water (L), Δt is the operation time (h) and A is the effective membrane area (1.93 × 10−4 m2).Water flux was measured using a dead-end filtration module at 0.2 MPa and 25 °C. Before recording the data, the membrane was pressurized for 30 min at 0.25 MPa. The water flux was measured every 15 min continuously for 180 min using the following equation:FRR) was calculated according to the following equation, where Jb is the water flux (L/m2h) of the initial membrane for BSA protein filtration and Jw is the water flux (L/m2h) of the washed membrane for pure water:The method for the BSA fouling experiment was the same as that for water filtration. Water was replaced by 1 g/L phosphate-buffered saline solution . The protein concentration of the permeation was measured by a UV-Vis spectrophotometer. After protein filtration, the used membranes were immersed in deionized water for 7 h, and the filtration cell was cleaned five times with water. Then, the filtration was started again. The flux rejection and recovery ratio  absorption bands at 1732 cm−1 should be attributed to the –C=O group in AMA. The characteristic peaks of SBMAA can also be observed at 1646, 1036 and 1209 cm−1, which is attributed to the symmetric and asymmetric stretch vibrations of –(SO3)2− groups, respectively.The structure of the P(AN-co-SBMAA-co-AMA) copolymers with pendant AMA groups is presented by an FT-IR spectrum in 1H-NMR spectra (3)=CH2). However, the spectra of the P(AN-co-SBMAA-co-AMA) copolymers did not show these signals. It should be noted that AMA has two different types of double bonds: conjugated methacrylic and unconjugated allyl groups. The reactivity of the methacrylic group is quite higher than that of the allyl group because of the presence of the electron-withdrawing ester group [3)3+ group in SBMAA, characteristic signals appear at 2.9 ppm. Allyl was introduced into P(AN-co-SBMAA-co-AMA) copolymers, and the reaction occured through the route shown in From the  spectra  of AMA, er group ,19. AllyThe membrane crosslinking density was evaluated by swelling, tensile strength and water pressure resistance tests. Swelling test results of three crosslinked membranes and the blank PAN membrane are shown in S) of the other crosslinked membranes CM-0.4, CM-0.8 and CM-1.2 decreased gradually. Furthermore, CM-1.2 retained almost the same shape as in the initial stage. However, the opposite tendency was observed for GF from Jw), BSA protein flux (Jb) and flux recovery ratio (FRR) of PAN-based membranes are illustrated in Jb) compared to that of the pure water flux (Jw). This means that small protein molecules existed in the membranes, which were deposited and adsorbed on the membrane surface. The CM-0.4, 0.8 and 1.2 membranes contained enough zwitterionic groups to result in a high flux-recovery ratio (FRR) even after three cycles of BSA solution ultrafiltration. An obvious reason is that the hydrophilic zwitterionic group, SBMA, with a balanced charge and minimized dipole, is an excellent candidate for nonfouling materials because of its strong hydration capacity owing to electrostatic interactions [The pure water flux (ractions ,21.In conclusion, crosslinked PAN membranes with different crosslinking densities were prepared by a facile thiol-ene click reaction under UV irradiation. By regulating the crosslinking densities, the membranes were presented with different topological structures. It was found that the mechanical properties of the membranes greatly improved after crosslinking. Moreover, the crosslinked membranes can maintain their shape well, even when soaped into DMF solution. It was also found that the introduction of betaine, a zwitterionic sulfonic acid, significantly improved the antifouling properties of the separation membranes. Therefore, crosslinked membranes can be used for high-pressure operation and for treating wastewater containing high concentrations of organic waste.


Ka of cytosine by more than 2 units , or the formation of non-specific PEG/DNA complexes appear to contribute insignificantly to i-motif stabilization. Molecular crowding, largely an excluded volume effect of added PEG, having a molecular weight in excess of 1,000 g/mol, appears to be responsible for stabilizing the more compact i-motif over the random coil at higher pH values.We have previously shown that c-MYC promoter sequences can form stable i-motifs in acidic solution (pH 4.5–5.5). In terms of drug targeting, the question is whether c-MYC promoter sequence i-motifs will exist in the nucleus at neutral pH. In this work, we have investigated the stability of a mutant c-MYC i-motif in solutions containing a molecular crowding agent. The crowded nuclear environment was modeled by the addition of up to 40% w/w polyethylene glycols having molecular weights up to 12,000 g/mol. CD and DSC were used to establish the presence and stability of c-MYC i-motifs in buffer solutions over the pH range 4 to 7. We have shown that the c-MYC i-motif can exist as a stable structure at pH values as high as 6.7 in crowded solutions. Generic dielectric constant effects, e.g., a shift in the p Our research group in particular has published G-quadruplex studies on these oncogenes which have demonstrated G-quadruplex- and i-motif-forming sequences in their promoter regions. Located on the human chromosome 8, the c-MYC gene is responsible for encoding a transcription factor that can regulate many genes that can further affect cell function, cell growth and cell apoptosis [1, is believed to control over 90% of the c-MYC gene transcription [1 is located ~142 to ~115 bp upstream of P1 promoter, and is capable of forming higher order DNA structures such as looped out G-quadruplexes and i-motifs due to its GC-rich rich sequence [Regulation of the expression of oncogenes such as K-Ras , Bcl-2  may provpoptosis . The c-Mpoptosis ,6. It ispoptosis ,8. The ccription ,13,14,15sequence ,17,18.Ka of cytosine N3 . In effect, the i-motif is most stable at the pH where one half of the cytosines are protonated and three H-bonds are formed between the two paired cytosines. While G-quadruplexes formed by G-rich sequences have received more attention since their discovery, studies of the i-motif formed by C-rich complementary strands have been rather limited. To date, most i-motif studies have focused on geometry, symmetry, folding topology and subsequent stability. Although most C-rich sequences (including the c-MYC promoter sequence) can form an ensemble of i-motifs depending on folding topology and the specific cytosines being paired, a typical structure for the c-MYC i-motif is shown in Intercalated motifs or i-motifs are formed by the unusual cytosine/cytosine base pairing shown in +-H / N3-Cyt pair [The mutant c-MYC promoter sequence, (5′-TTTTCCTACCCTTCCTACCCTAA-3′), used in this study was designed on the basis of previous studies, to yield a simple folding topology. In fact a single melting transition is observed for this sequence in DSC experiments. Computational modeling of the intramolecular i-motifs formed by a similar mutant c-MYC 23-mer P1 promoter sequence, (5′-CTTTCCTACCCTCCCTACCCTAA-3′), yielded an ensemble of five different possible i-motif structures, of which three were observed experimentally in DSC experiments . The strCyt pair ,27,28,29i.e., density, viscosity, and bulk dielectric constant. The fundamental difference between smaller co-solutes and these larger macromolecules is that the polymers have much larger excluded volume effects. The excluded volume affects the chemical and biochemical reactions happening in the cell [To date, most of the studies of i-motif structure and stability have been done on oligonucleotides in dilute solutions with little or no co-solute or molecular crowding agent present. However, the nuclear solution environment is anything but dilute. Macromolecules, e.g., DNA, various RNAs, and numerous proteins are present in the nucleus, with water accounting for only 60% of the solution mass in the nucleus ,31. The the cell . The crothe cell ,35,36,37the cell ,42,43,44the cell ,47,48,491 P1 promoter region, as a target for drug therapeutics, would depend on the existence of a stable i-motif structure in the nucleus at or near neutral pH. If stabilization of the P1 promoter sequence i-motif can be achieved, it would be possible to target the c-MYC or other oncogene promoter sequence i-motifs with drugs designed to form stable drug/i-motif complexes and thereby down-regulate the expression of these oncogenes.The efficacy of using i-motif forming sequences (C-rich sequences), located in the NHE IIIIn this work, we have investigated the potential for the formation of stable c-MYC i-motif structures at physiological pH in crowded environments. The question is whether a crowded environment favors the i-motif conformation over a random coil conformation for the DNA. We attempted to mimic the crowded nuclear environment by adding as much as 40% w/w polyethylene glycol to dilute solutions of the c-MYC C-rich oligonucleotide. The effects of crowding agent concentration and molecular weight have been explored in these studies. Polyethylene glycol (PEG) concentrations were varied from 10% to 40%, and the PEG molecular weight was varied from 200 g/mol to 12,000 g/mol. We present data that show the presence of the classical i-motif structure for the c-MYC P1 promoter sequence at pH values as high as 6.7. We have also shown that PEG effects are a mixture of co-solute and dielectric constant effects in combination with excluded volume or molecular crowding effects. All of these effects are linearly dependent on the PEG concentration while the molecular crowding effect is also dependent on the PEG molecular weight. The use of PEG as a molecular crowding agent is complicated by the fact that as the PEG molecular weight is increased above 4,000 and/or the PEG concentration is increased to 30 or 40% (w/w), PEG begins to interact with DNA (and probably RNA) forming non-specific DNA/PEG complexes. However, the results of this work still point to the fact that the c-MYC and other oncogene i-motifs are stabilized in crowded solution environments and therefore should be stable at neutral pH and present in the nucleus under physiological conditions.mTs) for the mutant c-MYC P1 promoter sequence i-motifs were first determined in both BPES (0% glycerol) and 20% w/w glycerol-containing solutions having pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0, using DSC. Theses mT data are shown in 2PO4, K2HPO4), 1 mM EDTA, and 100 mM KCl, BPES buffer, with and without added glycerol.The melting temperatures  is the result of a strengthened repulsive interaction between the two fully protonated cytosines (Cyt-H+/H+-Cyt). Alternatively, the stabilization at pH 5.5 is the result of a strengthened Cyt-H+/Cyt interaction between the two hemi-protonated cytosines at the higher pH.The mTs) for the c-MYC P1 promoter sequence i-motif in solutions containing PEG as the co-solute or molecular crowding agent, and at pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. In these experiments, the added PEGs had molecular weights that varied from 200 g/mol to 4,000 g/mol. In mTs at the seven specific pH values for the i-motif in BPES buffer and the change in the mT, ΔmT, brought about by the addition of 20% PEG .DSC was used to determine the melting temperatures , the mT measured is attributed to the unfolding of the i-motif while at higher pH values  the DNA samples that undergo thermal unfolding are not all classical i-motifs (see CD data below). The lowest pH data show that at pH 4.0, where the cytosine pairs are more completely protonated, the addition of glycerol or low molecular weight PEG  destabilizes the i-motif significantly (ΔmT ≈ −9 °C for PEG200). This destabilization is due to an increase in the electrostatic repulsion between charged cytosines, (Cyt-H+/H+-Cyt), brought about by the decrease in the dielectric constant for the water/PEG mixed solvent. The increase in i-motif stabilization at pH values above 4.5 exhibits a trend in that the stabilization of the i-motif is increased as the molecular weight of the PEG is increased, a clear indication of crowding due to the excluded volume effect of the larger polymers. In all of these solutions, there is a balancing act going on between the effect of the lowered dielectric constant  and the effect of molecular crowding (increasing with PEG molecular weight). Sugimoto’s group reported the same effect, an increase in i-motif melting temperature with higher molecular weight PEG co-solutes [The stabilization of the i-motif by the addition of PEG is evident from the increases in -solutes .mT, for the mutant c-MYC P1 promoter sequence i-motif without added PEG, and the increases in the mTs, ΔmTs, for the PEG8000 containing solutions are reported in The stabilization (and/or stability) of the c-MYC i-motif as a function of crowding agent concentration was again evaluated in DSC experiments. The melting (unfolding) temperature, mTs for the mutant human c-MYC i-motifs, particularly at higher pH values  in the presence of added PEG8000. The mTs reported in 8000, results in a 20 to 27 °C increase in mT at pH 6.0 or 6.5. This is a clear indication that the i-motif is stabilized in a crowded solution environment at pH values approaching neutral pH and that the i-motif could persist in the crowded nuclear environment.The data presented in 8000 containing solutions are shown in Typical DSC melting curves observed for the thermal unfolding of the c-MYC C-rich promoter sequence oligonucleotide obtained at pH = 5.0 in 20%, 30% and 40% w/w PEG8000 experiment (fit to a single two-state transition) shows only the melting of the i-motif. The DSC data for the 30% and 40% PEG8000 experiments (fit for two overlapping two-state transitions) show two separate effects upon the addition of PEG: 1) an increase in the mT for melting the i-motif (lower melting peak in these thermograms) and 2) a second peak with a higher melting temperature which is indicative of the presence of a small amount of a non-specific PEG/DNA complex. The blue solid lines and the red dotted lines in msT ≈ 50, 53, and 57 °C, are attributed to the unfolding of the i-motif in these solutions while the higher melting process, msT ≈ 62 and 65 °C are speculated to be the unfolding of a non-specific DNA/PEG complex formed in the 30% and 40% PEG8000 solutions. The assignment of the higher temperature melting to the DNA/PEG complex is based on its mT values which are much higher than mT values reported for unfolding of the c-MYC intramolecular i-motif [The raw DSC data  286 nm while the “i-motif-like” DNA structure exhibits a maximum ellipticity at (or near) 275 nm. CD spectra obtained for the mutant c-MYC i-motif in solutions with and without added glycerol are shown in 2000 drives the refolding of the i-motif into the classical structure at pH = 6.0. The addition of a molecular crowding agent, e.g., high molecular weight PEG, is apparently driving the folding/unfolding equilibrium toward formation of the more compact i-motif structure even though the cytosine pairs at this pH  are largely deprotonated.The CD spectrum at pH 4.5 in BPES buffer without added PEG is consistent with previously published reports in that the classical i-motif structure is observed in acidic solutions. The classic CD spectrum for the i-motif is shown as the black line in vs. pH for the c-MYC promoter sequence oligonucleotide in 0%, 20%, 30% or 40% PEG8000 is shown in The stabilization (and/or stability) of the c-MYC i-motif as a function of crowding agent concentration was evaluated in CD experiments. A plot of the molar ellipticity at 286 nm mT  to less than 14 as calculated from the Born approximation [Ka suggested by Sugimoto [Interestingly, DNA/PEG complexes have been reported in the literature as part of a biological technique that has been used to transport transgenic DNA across cell membranes ,55,56,5750 g/mol . As mentximation . Howeverximation ,59. Cleaet al. describes the stabilization of the human telomere G-quadruplex in PEG solutions to be solely the result of the non-specific interactions driving DNA/PEG complex formation [8000 and PEG10000 and at concentrations up to 42.5% (v/v). Although Buscaglia et al. discount molecular crowding in their study [2000, 20% PEG4000, and 20% PEG8000), at least by DSC.A more recent publication by Buscaglia ormation . They weir study , we obseC→T mutations and two C→A mutations. The purpose of these mutations was to simplify the folding and hopefully end up with a c-MYC promoter sequence that exhibited only a single i-motif folded conformer. The WT sequence has multiple repeated cytosines, which can interact to form more than one i-motif or “i-motif like” structure. In order to simplify the model, we designed Mut1 with the above listed 5 mutations and MutA with seven point mutations, including two additional C→T mutations. The sequences for the 5-base and 7-base mutants are listed in The mutant human c-MYC promoter i-motif forming sequence used in this study was designed based on the results of previous research. Previously we used a mutant sequence, Mut1, in which there were three 260 = 198,200 M−1cm−1.The lyophilized oligonucleotide for MutA was purchased from Midland Reagent, Inc. . The oligonucleotide was dissolved in 1 mL of each buffer to make a stock DNA solution. DNA concentrations in the stock solution were verified using a Olis HP 8452A Diode Array Spectrophotometer  and a molar extinction coefficient ε2PO4, 10 mM K2HPO4, 100 mM KCl, and 1 mM EDTA (for one liter of buffer). The solutions were filtered using a 500 mL Corning  bottle top filter immediately after being prepared. The pH values of each of the buffer solutions were adjusted to the correct pH by adding either NaOH or HCl and measured using an Accumet XL15 pH meter . BPES buffer solutions containing glycerol, and PEG at various molecule weights and concentrations were prepared by measuring the mass of certain amount of BPES buffer, then the mass of co-solute was added based on the desired concentration and adjusted to desired pH values by adding either NaOH or HCl.All chemicals used in this study were purchased from Sigma-Aldrich  and used without further purification. The BPES buffer used in this study was prepared using 10 mM KHTm) and the enthalpy change, ΔH, for the unfolding process for every DNA sample.DSC experiments were performed on a Microcal VP-DSC . The nominal concentration for DSC experiments was 300 μM. The DSC experiments were done over a temperature range from 10–90 °C at a scan rate of 90 °C/h. At minimal of three scans for each DSC experiment were acquired in order to ensure reproducibility. The data was analyzed with Origin 7  in order to determine the melting temperature (8000 and PEG12000 CD titration experiments), CD samples were also made up from the pre-DSC samples. The CD titration samples were extracted from the leftover pre-DSC samples for each PEG8000 containing buffer. The pH of each pre-DSC sample was corrected to 4.0 before making the CD samples. The nominal concentration for each CD sample was 3 μM. The CD spectra were collected at 20 °C over the wavelength range of 200 to 325 nm with constant N2 gas flow. Data was analyzed using Olis GlobalWorks and GraphPad Prism 5 .CD experiments were performed on an Olis DSM 20 CD spectropolarimeter . The CD samples were extracted from the post-DSC samples, which are collected after each DSC experiment. The nominal concentration for each CD sample was 3 μM. In some experiments . Samples were prepared for the DLS measurements by diluting the stock DNA solutions to a final same concentration of 3 µM ELP in BPES buffer with and without added PEG12000. These sample solutions were stored at 4 °C, and then immediately before a DLS measurement, 1.5 mL of the dilute solution was placed in a conical tube and spun for approximately 5 min using a TOMY HF-120 Capsulefuge  with an RCF of approximately 2000 G. Approximately 200 µL of the ELP sample was withdrawn from the conical centrifuge tube and used to overfill the 10 µL DLS quartz cuvette. All of these operations were done at room temperature. The DLS quartz cuvette was examined to ensure that no air bubbles were present in the active volume, and the cell allowed to equilibrate for 5 min after being placed into the DLS instrument. All DLS data was collected at 658 nm using a 10 s acquisition time. Sample collections were repeated for at least five times with five min intervals in between to ensure reproducibility. The laser power was set to 50%, the auto attenuation mode was disabled, and the translational diffusion value, Dt, were recorded. The data was analyzed using the DYNAMICS software package  included with the instrument.Dynamic light scattering experiments were performed using a DynaProIn this work, we probed the influence of both low molecular weight co-solutes and high molecular weight crowding agents on the stability of the c-MYC C-rich promoter sequence i-motif in crowded environments at pH values approaching neutral pH. Low molecular weight co-solutes included glycerol and 200 and 400 g/mol PEG. The crowded environment of the nucleus was simulated by higher molecular weight PEGs at concentrations as high as 40% w/w. We were able to demonstrate that the smaller co-solutes had the effect of improving charge stabilization through both a reduction in the water activity and a decrease in solvent dielectric properties.8000, the i-motif was found to be stable in solutions having pH values as high as 6.7, fairly close the pH = 7.0 we were trying to achieve. We found that stabilization of the DNA i-motif by PEG, especially for higher molecular weight PEGs , was offset to some degree by the non-specific interactions between the DNA random coil and the higher molecular weight PEGs. The best result we achieved, in terms of i-motif stabilization, was for the mutant c-MYC C-rich promoter sequence in 30% w/w PEG8000 solution where a stable i-motif structure was observed at pH values as high as 6.7. In these solutions, the i-motif still had a characteristic CD signal and a melting temperature in excess of 38 °C. Unfortunately, once the concentration of PEG8000 is increased to 40% w/w or more, or the PEG molecular weight is increased beyond 8,000 g/mol, the formation of DNA/PEG complexes results in a decrease in the fraction of the DNA existing as an i-motif and a concomitant increase in the fraction of the DNA present in the form of a DNA random coil complexed with PEG.Although not measured here, the volume of folded i-motif is estimated to be smaller than the volume of the DNA random coil. In effect, the i-motif to coil transition requires greater energy in these crowded solutions to unfold the DNA than would be needed in water. We started this work with the idea of answering just one question, can an i-motif exist as a stable structure at neutral pH. In the presence of 30% w/w PEGi.e., their effect on water activity and solvent dielectric constant, contribute only negligibly to i-motif stabilization at higher pH values. It is also clear that the formation of non-specific random coil DNA/PEG complexes would appear as destabilizing in terms of competing with the formation of i-motif DNA species. In comparison, the molecular crowding (or excluded volume effects) of higher the molecular weight co-solutes  has been shown to contribute significantly to the stability of the more compact i-motif structure relative to the larger random coil DNA. Even though we achieved stabilization of the i-motif to pH = 6.7, we found that the use of the uncharged high molecular weight PEGs to model the crowded nuclear environment is problematic since these polymers associate with random coil DNA by hydrogen bonding [Nevertheless, we predict that stable i-motif structures can exist at neutral pH under the crowded solution conditions in the nucleus. It is clear from the data presented here that the stabilizing effects of low molecular co-solutes,  bonding . Althoug


Rhus typhina L.) fruits. Initially, influencing factors such as liquid-solid ratio, duration of ultrasonic assisted extraction, pH range, extraction temperature and ethanol concentration were identified using single-factor experiments. Then, with respect to the three most significant influencing factors, the extraction process focusing on the DPPH· scavenging capacity of antioxidants was optimized using RSM. Results showed that the optimal conditions for antioxidant extraction were 13.03:1 (mL/g) liquid-solid ratio, 16.86 min extraction time and 40.51% (v/v) ethanol, and the desirability was 0.681. The UPLC-ESI-MS analysis results revealed eleven kinds of phenolic compounds, including four major rare anthocyanins, among the antioxidants. All these results suggest that UAE is efficient at extracting antioxidants and has the potential to be used in industry for this purpose.For the first time, response surface methodology (RSM) using a Box-Behnken Design (BBD) was employed to optimize the conditions for ultrasonic assisted extraction (UAE) of antioxidants from Chinese sumac ( Rhus typhina L.) is a perennial and flowering shrub that belongs to the genus Rhus. It originally came from Canada and the United States and is widely distributed in the Northern Hemisphere’s subtropical to temperate regions −, and were tentatively identified as two isomers of glucocaffeic acid. Peaks 4, 5 and 6 showed deprotonated molecular ions of m/z 301.0, m/z 447.0 and m/z 301.0 respectively, and were identified as ellagic acid, quercetin-3-rhamnoside and quercetin, respectively. From the UPLC total ion chromatogram - β-d-galactoside; this is a rare 7-O-methyl cyanidin compound as proven from the NMR spectrum + = 433 Da + = 433 Da. Peak 10 was tentatively identified as 7-O-methylcyanidin-3-O- -β-d-galactopyranosyl-4-vinylcatechol, which is proposed for the first time. Although it has been found that the degradation of active ingredients may occur during application of UAE in food processing [It was found that the composition of the Chinese sumac reported here was similar to that of according to previously published information . Peaks 2matogram B, it wasocessing ,14, the Staghorn sumac fruits were harvested in September 2013 in Beijing, China. 1,1-Diphenyl- 2-picrylhydrazyl (DPPH) radical reagent was obtained from Sigma . All other chemical reagents used were of analytical grade.Gentle rubbing by hand was used to collect the staghorn sumac fruits from the staghorn plant. Then about 200 g of the collected fruits were freeze dried  and ground with a blender, before being filtered through 40 mesh sieve. The powder (1.0 g) was then mixed with ethanol of different concentrations to form the required liquid-solid ratios. Then the mixture was adjusted to different pH concentrations and extracted using the JCX-100G ultrasonic instrument . The extract was centrifuged  at 10,000 rpm for 10 min at room temperature, after which the supernatant was filtered through a 0.2-μm PTFE membrane filter  in preparation for spectrophotometric and UPLC-MS analyses. All procedures were conducted under reduced light levels, and all experiments were conducted three times and an average was obtained.0 is the absorbance of DPPH alone, A1 is the absorbance of DPPH + extract and AS is the absorbance of the extract only. All samples were tested in triplicate.The DPPH radical scavenging capacity assay was based on a previously described method  with somInitially, the appropriate ranges and influencing factors were determined in a preliminary study so that they could be used to design the main experiments. Many factors including extraction time, composition of solvent and solvent to solid ratio, could affect the UAE extraction efficiency. All of these factors were evaluated to identify the most influential factors when trying to obtain the maximum possible extraction of the antioxidant compounds when using UAE.RSM was employed to establish the optimum conditions for extracting antioxidants from Chinese sumac ,17. The m/z.The chromatographic analyses of the extracts were performed on a UPLC Acquity chromatograph coupled with a TQD Acquity mass spectrometer , which had an electrospray ionization (ESI) source. A C18 BEH Waters Acquity column  was used [p < 0.05 was considered to be a significant difference. The analyses of the RSM data were done using Design-Expert 7.1 .The analyses of the data were done using SPSS v19.0 statistical package . The experimental data were subjected to χ 2 tests, and The study represents the first report on the feasibility of ultrasound-assisted extraction for the maximizing recovery of antioxidants from Chinese sumac. RSM was used to investigate the main and interaction effects of important independent variables for extraction of antioxidants on the basis of single-factor experiments. Ultrasound-assisted extraction with acidified ethanol was proved to be efficient technique for easy and rapid isolation of high yields of antioxidants from Chinese sumac at room temperature (20 °C). The optimal conditions for antioxidants extraction were 13.03:1 (mL/g) liquid-solid ratio, 16.86 min extraction time and 40.51% (v/v) ethanol, and the desirability was 0.681. In addition, a total of 11 phytochemicals including phenolic acids, flavonoids and anthocyanins were identified from the antioxidants by UPLC-MS. Pyranoanthocyanins are thought to contribute to the orange hues observed during wine maturation and aging and have higher antioxidant potential . Therefo


Human Immunodeficiency Virus (HIV) and Tuberculosis (TB) are the double burden diseases of the world. The African continent takes a great share of TB-HIV cases worldwide. This study was aimed to determine the prevalence of TB-HIV co-infection in Ethiopia, using a meta-analysis based on a systematic review of published articles.An electronic search was conducted in databases including PubMed, HINARI, EMBASE, Cochrane library and Google Scholar to extract the articles. Articles published between 1995 and November 2017 had been searched for using different keywords. The analysis was performed using MetaXL software and R statistical software (version 3.2.3).n = 26,746) records from 30 articles of which 21 were cross-sectional, 7 were retrospectives and 2 were prospective studies. The range of prevalence of TB-HIV co-infection was found to be from 6 to 52.1% with random effects pooled prevalence of 22% (95% CI 19–24%) and with substantial heterogeneity chi-square (X2) = 746.0, p < 0.001, (I2 = 95.84%).Our searches returned a total of (Our analysis indicated that the prevalence of TB-HIV co-infection is high in Ethiopia with substantial regional variation. An integrated, facility-based and community-based effort towards the prevention, early detection and management of cases should be further strengthened throughout the country to mitigate the double burden disease. Human Immunodeficiency Virus (HIV) remained as the leading cause of mortality and morbidity; in 2016 for instance, 1.8 million people were newly infected and 1 million AIDS-related deaths were registered . Hence, The burden of TB is still alarming due to the emergences of drug-resistant strains. Recent studies indicated that the prevalence of MDR-TB in Ethiopia is particularly noticeable 15%) in previously treated TB cases % in prev. This inIn general, people living with HIV are 20 times riskier to be infected with TB as compared to HIV negative people , with caIn high burden and resource-limited countries like Ethiopia, integrating health services on TB and HIV has paramount importance in order to increase the effectiveness of the diagnosis and to improve the management approaches . ConsideAn electronic search was conducted to retrieve and recruit studies. Published articles of cross-sectional, prospective and retrospective cohort studies were included, which focused on the “Prevalence of TB-HIV co-infection in Ethiopia”. Databases PubMed, HINARI, EMBASE, Cochrane library, and Google Scholar search engine were used to extract journals. Articles published between 1995 and November 2017 had been searched for and their full-text was retrieved by two independent reviewers.n > 100). 3) Studies written in English. 4) Articles which were published and available online.The predetermined inclusion criteria for this analysis were: 1) studies conducted in Ethiopia, 2) either longitudinal/cohort or cross-sectional studies which assessed the prevalence of TB/HIV co-infection in Ethiopia and has a sample size of greater than 100 . When working on binary data with low prevalence, the inverse variance weight in fixed-effects meta-analyses is sub-optimum. Hence, the estimated prevalence was determined using the variance stabilizing double arcsine transformation . Wilson ogeneity –26. The iability . To analiability  and by an = 26,746) records from 30 articles of which 21 were cross-sectional, 7 were retrospectives and 2 were prospective studies. Furthermore, 17 studies had more than 500 participants; 12 studies were conducted in the Amhara region, 6 in southern Ethiopia, 2 in Tigray, 3 in Oromia, 3 in Addis Ababa, 2 in Afar and 2 across Ethiopia.Our searches returned a total of  and 11 studies determined TB among HIV/AIDS patients (5370) (see Table Nineteen  studies 2) = 746.0, p = 0.0001, (I2 = 95.85% 95% CI: 94.9–96.6)  Fig. .Fig. 2ThAs part of our sensitivity analyses, we excluded one large study ; estimatThis systematic review and meta-analysis of TB-HIV co-infection identified 30 studies of 26,746 individuals in Ethiopia. Our pooled results confirmed that the prevalence of TB-HIV co- infection is 22% in Ethiopia. Furthermore, the high prevalence of HIV/AIDS in the general population is related to a higher prevalence of TB-HIV co-infection. This result is almost consistent with one global study  but highAccording to the subgroup analysis, the highest prevalence was found in the Addis Ababa region (31%), followed by the Afar regional state (29%), whereas the lowest prevalence was found in southern Ethiopia (18%). The substantial difference between the regions might be due to the difference in the socio-cultural and demographic characteristics of the population and associated HIV prevalence , 64. SupAs Ethiopia is among the high burden countries for TB and HIV, it may be expected to find a high prevalence of TB-HIV co-infection. However, the pooled prevalence of TB-HIV co-infection (22%) is still higher than the WHO estimated figure (8%) in 2017 . The pooTB-HIV co-infection has a great impact on the health outcome of the victims due to accelerated progression. Co-infected individuals have significantly lower years of survival as compared to those HIV positive only . WitnessOur analysis indicated that the prevalence of TB-HIV co-infection is high in Ethiopia with substantial regional variation. Therefore, an integrated and consistent facility-based as well as community-based effort addressing the prevention, early detection and management of TB and HIV infection should be further strengthened throughout the country to mitigate the double burden disease.


AP-PRP). Autocrosslinked hyaluronic acid (AHA) was prepared by organocatalyzed auto-esterification and structured in microparticles (MPAHA) by shearing. The attenuation of the negative charge of MPAHA was performed with chitosan (CHT) by polyelectrolyte complexation yielding MPAHA-CHT. The results showed that microparticles (MPs) have viscoelastic properties, extrusion force and swelling ratio appropriate for injectable applications. The association of AP-PRP with the controlled structure of MPAHA and MPAHA-CHT formed a matrix composed of platelets and of a fibrin network with fibers around 160 nm located preferably on the surface of the MPs with an average diameter of 250 μm. Moreover, AP-PRP/MPAHA and AP-PRP/MPAHA-CHT associations were non-toxic and supported controlled growth factor (PDGF-AB and TGF-β1) release and in vitro proliferation of h-AdMSC with a similar pattern to that of AP-PRP alone. The best h-AdMSC proliferation was obtained with the AP-PRP/MPAHA-CHT75:25 indicating that the charge attenuation improved the cell proliferation. Thus, the association of AP-PRP with the controlled structure of HA can be a valuable approach for orthopedic applications.Platelet-rich plasma (PRP) associated with high molecular weight hyaluronic acid (HA) has been clinically used for tissue regeneration in orthopedics. Despite the recognized beneficial clinical outcomes , its use is still challenging and controversial due to lack of standardization of association practical protocols. Moreover, most studies neglect the matrix structure, that generates the ultimate properties of the association among platelets, fibrin network and the microparticles. In the present work, we aimed to analyze the influence of the PRP/HA association with a controlled matrix structure on the stability, rheological behavior, release of growth factors and in vitro proliferation of human adipose-derived mesenchymal cells (h-AdMSCs). The attenuation of the negative charge of HA was also evaluated. Pure PRP (P-PRP)  was prepared by centrifugation and activated with serum and calcium chloride ( TMore recently, PRP has been associated with high molecular weight hyaluronic acid (HA), mainly for orthopedic therapies ,5. HA isAlthough several clinical reports have shown that the association of PRP and HA promotes tissue regeneration, improves the mobility of patients, relieves pain and reduces the risk of infection in orthopedics treatment, studies demonstrating its effectiveness are still limited and controversial ,9. Most Moreover, the standardization and optimization of this association , molar mass of HA, need for crosslinking of HA, the structure of HA , and protocols of mixture of PRP and HA, are still a challenge. Despite the numerous studies described in the literature, as far as we know, there is only one study associating PRP/HA-based on a matrix structure  formed bThus, this study, which also considers the structure of the matrix (platelets + fibrin network + microparticles), is the starting point to better understand the complex processes involved in this association and in tissue healing. The crosslinking of HA prevents the rapid degradation of HA chains by enzymatic action and/or by reactive oxidative species (ROS), and to extends the action over time. Moreover, the crosslinking improves the mechanical properties, such as dumping and viscoelasticity of HA, when compared to the non-crosslinked one. The structuring of microparticles enables the increase of the surface area for cell spreading and adhesion, contributing to the proliferation and control of matrix structure. Moreover, the reduction of the negative charge by the addition of chitosan, improves cell adhesion on the microparticles, while the fibrin network from activated PRP acts as the natural scaffold favoring the cell adhesion and proliferation, and improving the mechanical properties and the stability of matrix (HA microparticles + fibrin network).AP-PRP/HABDDE structured in microparticles has benefited the physicochemical and mechanical properties compared to FHA, the release of growth factors was similar to fibrin network from AP-PRP and the mesenchymal cells proliferation (which are progenitors for bone and cartilage mature cells contributing to bone and cartilage repair). However, Shimojo et al. used 1,4-butanediol diglycidyl ether (BDDE) as crosslinkers . AlthougAP-PRP) with auto-crosslinked HA microparticles (MPAHA). Ren et al. [Our hypothesis is that a controlled matrix structure will increase the stability of association P-PRP/HA preventing phase separation of free HA and will enable standardization of protocols for clinical applications. Thus, the present work aimed to analyze the influence of the P-PRP/HA association with controlled matrix structure on the stability, rheological behavior, delivery of growth factors and in vitro proliferation of human-adipose mesenchymal cells (h-AdMSCs). The control of matrix structure was obtained by association of fibrin network from activated P-PRP  was obtained from Spec-Chem Ind. . Chitosan  was purchased from Polymar®  and purified according to a protocol described by Nasti et al. [Hyaluronic acid (HA) in sodium salt form  for 8 h to form hyaluronic acid . The suspension was centrifuged in a Rotina 380R centrifuge  at 10,000 rpm for 10 min to remove the resin, and it was then neutralized with tetrabutylammonium hydroxide (TBA-OH) 0.2 mol/L  to form the quaternary ammonium salt of HA . The solution was then frozen and lyophilized in a Liobras L101  for 48 h. Initially, in order to prepare AHA, the intermediary tetrabutylammonium hyaluronate (HA-TBA) was prepared. In brief, an aqueous solution of 0.5% (g/100 mL) sodium hyaluronate  was stirred in strongly acidic ion exchange resin DowexAHA was prepared by esterification reaction organocatalyzed with trimethylamine  accordin−1 with a resolution of 4 cm−1 over 64 scans. Chemical characterization of AHA was performed by Fourier transform infrared (FTIR). Measurements were performed in a Thermo Scientific Nicolet model 6700 , in the ATR mode with accessory SMART OMNI-SAMPLER, in the spectral range of 4000–675 cmMPAHA microparticles were obtained by high shear homogenization in an Ultra-Turrax T25 homogenizer  at 18,000 rpm for 20 min of plane hydrogel swelling in ultrapure water. MPAHA were characterized by effective crosslink density Flory-Rehner calculations, average diameter, degradation time, rheology, extrusion force, swelling ratio, and cytotoxicity.Effective crosslink density Flory-Rehner calculations. Effective crosslink density was evaluated by measuring the volumetric swelling and using a simplified version of the Flory and Rehner equation. The value used for chitosan-water interaction parameter (χ) was 0.439 [as 0.439 .Degradation time. The gravimetric method described by Tang et al. [g et al.  was usedAverage diameter. The average diameter of MPs was measured in water. The measurements were performed with Malvern Mastersizer S laser diffraction . The standard deviation was calculated from ten consecutive measurements.Rheology. Rheological measurements were performed in the oscillatory and steady regime at 25 °C using a parallel plate geometry of 20 mm. Oscillatory measurements were conducted in the linear region with a stress of 1.188 Pa and in the frequency range of 0.1 to 10 Hz. Steady shear measurements were carried out at shear rates of 0.1–50 s−1. All rheological measurements were performed on a rheometer Haake RheoStress 1 . The parameters were calculated from the rheological curves, such as complex viscosity (G*), tan δ and power-law index (n) .Extrusion force. The MPs were firstly loaded in 1-mL plastic syringes with 30-gauge needles. Afterward, the force required to extrude was measured in an MTS 810 Servo-hydraulic Universal Testing Machine  (Load Cell 1.5 kN) at 25 °C at a 5.0 mm/min extrusion rate.Swelling ratio. Swelling measurements of the MPs were performed in PBS at 37°C for 72 h, according to Shu et al. [u et al. . The sweCytotoxicity. The cytotoxicity of the MPs expressed as the viability of h-AdMSCs cells was carried out by exposing them to cell culture at 37 °C for 24 h (ISO 10993-5: "Tests for Cytotoxicity—In Vitro Methods"). Then, cell viability was evaluated using the MTT assay  , according to the modified Mosmann method [n method . 3+) of CHT (pKb ~ 6.5) and carboxylic groups (COO−) of AHA (pKa ~ 2.9) [w/w). The reaction mixture at room temperature was kept under stirring at 1,000 rpm overnight. Then, the formed precipitates were washed with water and phosphate buffer saline (PBS) to pH 7 (pH 6.6–6.8). The mass ratio AHA:CHT used in the microparticles were 90:10 , 75:25 , and 50:50 . The charge ratios (R+/-) were estimated using the mass ratios AHA-CHT of MPs according to modified model by Rädler et al. [The attenuation of negative charges of AHA was performed by chitosan (CHT) addition. Polyelectrolyte complexes of AHA-CHT  were proa ~ 2.9) . Brieflyr et al. . ChemicaMPAHA-CHT in different mass ratios were obtained by high-speed shearing in an Ultra-Turrax T25 homogenizer  at 18,000 rpm for 20 min of plane hydrogel swelling in ultrapure water. MPAHA-CHT were characterized as described in et al. [Plasma rich in platelets and poor in leukocytes (P-PRP) was prepared according to Perez et al. . P-PRP was prepared from the whole blood (WB) of donors, who were healthy individuals aged between 30 and 40 years old and previously assessed through clinical examination. Human adipose tissue-derived mesenchymal stem cells (h-AdMSCs) were provided by the Umbilical Cord Blood Bank of Haematology and Hemotherapy Center of the University of Campinas. This research was approved by the Ethics Committee of the Medical Sciences School of the University of Campinas .® Zentrifugen, Tuttlingen, Germany) at 100× g for 10 min at 25°C and the upper layer was collected to obtain P-PRP [3) and in P-PRP were determined using the ABX Micros ES 60 hematology analyzer . P-PRP was obtained with approximately twice the platelet concentration (495.000 ± 18.810 pq/mm3).Briefly, whole blood (WB) was collected into 3.5 mL vacuum tubes  containing sodium citrate 3.2% (g/100 mL) as an anticoagulant. WB was firstly centrifuged in a Rotina 380R centrifuge (Hettichin P-PRP . The con2 solution (10% g/100 mL) as agonists applying the following conditions: agonist/P-PRP 20% (mL/100 mL), Serum/CaCl2 volumetric ratio 9. These proportions provide fibrin network architecture with thin fibers (approximately 160 nm average radius), which favors the proliferation of h-AdMSCs due to its paracrine nature. Serum was prepared by collecting 5 mL of WB in tubes without anticoagulant. After waiting 30 min to allow clot formation, WB was centrifuged at 2000× g for 10 min. The preparation and activation protocols followed Perez et al. [The activation of P-PRP was done with serum-containing thrombin and CaClz et al. ,34. 2) for 5 min. Then, the association between AP-PRP and MPAHA (and MPAHA-CHT) was performed in laminar flow cabinet by adding freshly activated P-PRP to MPs, in a volume/mass ratio AP-PRP:MP of 200 µL:200 mg. The components were mixed with the tip of a pipette for a short time in 48-well microplates and kept for 45 min for gel structuring. DMEM-LG  was then added to the wells for cell culture. MPs were sterilized by steam in the autoclave at 126 °C  and transforming growth factor β1 (TGF-β1) was performed after 45 min of gelation of AP-PRP alone, AP-PRP/MPAHA and AP-PRP/MPAHA-CHT in the presence of the Dulbecco′s Modified Eagle′s Medium with a low glucose concentration  in 48-well microplates. The microplates were kept in an incubator with 5% CO2 over the assays. After sampling, the volume was replaced with fresh medium at 3, 6, 12, 48, and 72 h without removing the gels from the wells. The samples were stored at −80°C until all have been collected. The concentrations of the released GFs were measured using enzyme-linked immunosorbent assay (ELISA) kits , according to the manufacturer′s instructions and specifications.Culture of seeded h-AdMSCs. The pre-cultured h-AdMSCs were trypsinized and resuspended in P-PRP to a final cell concentration of 1 × 104 cells/mL. P-PRP containing h-AdMSCs was activated and immediately added to MPAHA or MPAHA-CHT in 48-well microplates. The association with seeded h-AdMSCs was kept at room temperature for 45 min for consolidation of the structure. After that, DMEM was added. The cultivation of h-AdMSCs was carried out in 48-well tissue culture plates by adding 1 mL of the culture medium DMEM-LG to the wells. The assays were done in quadruplicate. The culture was maintained at 37 °C over 10 days. Cultivations in AP-PRP alone were used as control. Cell proliferation was quantified by MTT assay. At 2, 4, 7, and 10 days of cultivation, the samples were removed and transferred to 24-well plates. MTT (1 mL of 1 mg/mL) was then added, and the cultivation proceeded at 37 °C for 4 h. The MTT solution was then discarded, and 1 mL of dimethylsulfoxide (DMSO) was added to dissolve the purple formazan crystals. The samples were shaken at 120 rpm for 30 min to ensure the homogeneous dissolution of the formazan dye, and 200 µL of each sample was then transferred to a 96-well plate. The optical density was measured at 595 nm using a microplate reader . The cell number in each well was quantified by a calibration curve of absorbance measured at 595 nm and the cell concentration previously constructed. Images of the structured gel. Images of the gel structures were obtained by scanning electron microscopy (SEM) 4 days after h-AdMSC cultivation. The samples were fixed in a solution of paraformaldehyde/glutaraldehyde for 2 h and then dehydrated in an ethanol series for 15-min intervals. The samples were dried in a critical point dryer BAL-TEC CPD 030 . After gold coating in a Sputter Coater POLARON, SC7620 , the samples were visualized in a scanning electron microscope Leo440i  with an accelerating voltage of 20 kV.p-value ≤ 0.05.Each experiment was carried out in triplicate unless otherwise specified. All results are presented as the mean ± standard deviation (SD). The experimental data from all the studies were analyzed using analysis of variance (ANOVA). Statistical significance was set to −1 attributed to O–H stretching vibration and between 3000 and 2800 cm−1 attributed to the C–H stretching vibrations and bands of N–H stretching vibrations overlapped with the O–H bands, as expected. FTIR spectroscopy was used to confirm the formation of AHA and AHA-CHT. The spectrum of AHA showed the characteristic bands of polysaccharides  between −1, which is attributed to the stretching vibration of C=O of carboxylic acyl groups (~1700 cm−1) and ester groups (1780–1760 cm−1). The crosslinking was characterized by a decrease in the peaks at the region of 1800–1700 cm−1 which was attributed to overlapping of the carbonyl and amide vibrations. The peak at 1598 cm−1 assigned to the anti-symmetrical stretching vibration of the carbonyl group of the carboxylate in the AHA appears slightly shifted to the left in the AHA-CHT, indicating the ionic interactions between the carboxylate group of AHA and the NH3+ group of CHT. Moreover, in the AHA-CHT spectrum a slight displacement of peaks was also observed, attributed to the symmetric stretch vibration of the carboxylate group νs (COO−) of AHA from 1407 to 1413 cm−1. The attributions of the absorption bands of AHA-CHT were performed according to Coimbra et al. . The AHA3+ between 1625–1560 and 1550–1505 cm−1, respectively [Due to the overlapping, it is difficult to distinguish any sign between the asymmetric and symmetric bending vibrations of the NHectively .−1, and in the peaks, 1374 cm−1 compared to CHT spectra. These changes also suggested the formation of AHA-CHT.The AHA-CHT spectrum also showed a ratio change in the peaks at 1037 and 1077 cme), molar mass between crosslinks (Mc) and average particle diameter were determined for MPs (MPAHA and MPAHA-CHT) and results are shown in The swelling ratio (SR), effective crosslink density (Vp < 0.05) the swelling properties compared to MPAHA due to the decrease of hydrophilic groups that make hydrogen bonds with water, confirming the ionic crosslinking. Moreover, SR of MPAHA-CHT90:10 was significantly higher than other mass ratios indicating the lowest crosslinking degree, as expected. Cells generally show good spreading, proliferation and differentiation on hydrophilic surfaces. Both HA and CHT have an abundant number of hydrophilic groups, such as hydroxyl, amino, and carboxyl groups, which promote water uptake in their structure. The addition of CHT significantly affected (* e) and the average molecular weight between crosslinks (Mc) which influence mainly the swelling, mechanical, rheological properties and the stability of hydrogels. The addition of CHT increased Ve, as expected. Moreover, we observed lower Mc values, indicating higher entanglement of chains.Flory & Rehner equations were used to determine the effective crosslinking density  [Particle sizes of <700 μm) .MPAHA-CHT50:50 shows 14% of weight loss while MPAHA shows 34%, confirming the physical crosslinking.The stability results a, expresMPAHA-CHT90:10, the other MPAHA-CHT were stable (weight loss < 20%) over the 14 days of the assay, showing a capacity to absorb large quantities of PBS without undergoing dissolution.Moreover, except for the As seen in MPAHA-CHT90:10 showed a similar degradation profile of MPAHA. The degradation rate was decreased by the addition of CHT, while the MPAHA-CHT50:50 proportion as a consequence of the highest crosslinking degree. The extrusion force increased with chitosan concentration reaching a maximum at the MPAHA and MPAHA-CHT determined by oscillatory and steady measurements are shown in MPAHA becomes more resistant to deformation and has a lower fluidity evidenced by tan δ values (damping) with the addition of CHT. Moreover, MPAHA and MPAHA-CHT exhibited behavior typical of so-called weak gels with tan δ (G”/G’) > 0.1  > 0.1 , regardlMPAHA-CHT showed G’ values larger than 700 Pa in 1 Hz, which was outside the adequate range (100–700 Pa) for injectable applications. Therefore, MPAHA-CHT should be dispersed in the fluid phase or have the crosslinking degree decreased to fit the required range. The flow curve  was not significantly affected , which were higher for the associations than those of AP-PRP even with the dilution effect. The G* value indicated AP-PRP/MPAHA-CHT50:50 (G* 185.5 Pa) renders the association more resistant to deformation due to the strong electrostatic interaction . The values of tan δ of the associations prepared from AHA-CHT were lower than AP-PRP/MPAHA and AP-PRP, but they were similar to each other.The interactions between AP-PRP as the fluid phase in a 1:1 volumetric ratio AP-PRP/MPAHA-CHT and MPAHA-CHT maintained the weak gel-type behavior  from AP-PRP/MPAHA and AP-PRP/MPAHA-CHT structures were approximately 70% and from the AP-PRP was 83%, while the release of TGF-β1 from the AP-PRP, AP-PRP/MPAHA and AP-PRP/MPAHA-CHT structures ranged from 58 to 70% in 12 h. Maximum TGF-β1 release was not observed until 72 h were reached. The release of PDGF-AB from the AP-PRP/MPAHA-CHT slowed down the release of PDGF-AB compared to AP-PRP alone  at physiological pH, they will have a positive charge that ionically interacts with acidic species . Thus, t release .AP-PRP/ MPAHA-CHT was governed by the degradation rate because the ionic crosslinking reduced the number of exposed negative charges, thereby compromising the electrostatic interactions. In contrast, the release from +, CD73+, CD90+, CD105+, and HLA-ABC+ or the absence of HLA-DR+, and CD34-, CD45- by flow cytometry (data not shown).To evaluate cell proliferation, the purified population of h-AdMSC was firstly characterized by immunophenotyping cell surface markers such as CD29AP-PRP/MPAHA and AP-PRP/MPAHA-CHT. The cell proliferation, measured by absorbance at 540 nm, was proportional to the cell concentration. AP-PRP was used as control.MTT assay quantified the proliferation of the h-AdMSCs in the associations AP-PRP/MP and AP-PRP, and the micrographs of 4 cells/well/platelet number) in all associations, meaning that the cells maintained in the matrices retained their viability. The results also demonstrate that the associations provided favorable microenvironments to support the cultivation and proliferation of h-AdMSC in vitro cultures because in all associations the cell viabilities were remarkable up to 10 days.The cell number per well per platelet number determined after two days exceeded the number of seeded cells  similar to AP-PRP after 10 days of cultivation. Except in AP-PRP/MPAHA, AP-PRP/MPAHA-CHT90:10, and AP-PRP/MPAHA-CHT75:25 the cell number determined after two days (3.5 × 104 cells/ well/platelet number) exceeded the number of seeded cells (~ 1 × 104 cells/well/platelet number), the data show a long adaptation phase, or lag time with incipient proliferation up to seven days, which was attributed to the negative charge of the associations. On the 10th day, we observed an abrupt cell growth for AP-PRP/MPAHA-CHT75:25 with the cell number statistically (*p < 0.05) similar to AP-PRP.Although for AP-PRP and AP-PRP/MPAHA-CHT50:50 showed an exponential growth after two days confirming the influence of the negative charge attenuation on cell cultivation. These results suggest that CHT coverage favored h-AdMSC proliferation and the negative charge attenuation reduced the lag-phase time.AP-PRP) AP-PRP. AP-PRP) even with the dilution effects of 1:1 in the associations (200 μL of AP-PRP:200 μg) of MPs. Therefore, the reduction of negative charge density of the microparticles favored cell proliferation. The possibility to obtain injectable formulations opens perspectives on the use of these biomaterials in non-surgical applications of regenerative medicine, especially in orthopedics.In this work, we studied a matrix consisted of platelets, fibrin network  and HA crosslinked microparticles. We considered the stability, the rheological properties, the toxicity, the superficial area and the superficial charge of the matrix as the critical properties for the ‘association’ to be successful. These properties were evaluated and the results showed that the matrix is promising for orthopedic tissue engineering. The auto-crosslinked microparticles approach represents a strategy for controlling the hyaluronic acid structure for association with P-PRP . This approach is safe, allows easier protocol standardization and may be useful for clinical applications. Moreover, the microparticles improved the physicochemical and mechanical properties of activated P-PRP and provided a large surface area for cell adhesion and proliferation. The association formed matrix (microparticles + fibrin network + platelets) with the fibrin network preferentially on the surface of the microparticles. The coverage with chitosan attenuated the negative charge of the microparticles, producing proliferation profiles similar to the activated P-PRP alone (200 μL of